ABSTRACT:

Significant new advances at the molecular level in the field of plant–biotic interactions have led to an expansion in the range of potential strategies for genetically engineered resistance in crops during the past years. Nevertheless, the evolution of virulence by pathogens and pesticide resistance by insects represents continuing challenges in agriculture that have impelled a constant interest for deeper understanding of the molecular bases of plant-biotic interactions. The aim of this proposal is to investigate molecular mechanisms related to plant responses to biotic stresses, with emphasis on regulatory networks involved in plant-biotic interactions that lead to disease or to resistance. The framework of our proposal is founded on the need to advance our knowledge on the molecular and functional basis of biotic interactions among plants, pathogens and insects economically relevant to the Brazilian agriculture and to foster effective collaborations under a multidisciplinary context that are likely to contribute with procedures best suited for collecting pertinent data in a most efficient way. The specific goals of the research proposed are:

1) To identify host partners (co-factors and regulators) of geminivirus movement proteins. The identification of NSP partners from tomato and Arabidopsis has been successfully conducted in our laboratory using yeast two-hybrid screens. This has led to relevant scientific achievements including, for example, the identification of a novel antiviral signaling pathway (see below) and the first identification of a GTPase protein that facilitates translocation of NSP from the nucleus to the cytoplasm. A major perspective will be to determine the cellular function of NIG (NSP-interacting GTPase) and to examine its interaction with the nucleocytoplasmic transport machinery. For the case of the movement protein (MP), which facilitates the cell-to-cell movement of viral DNA, we will examine whether MP accomplishes its tasks through interaction with host proteins.

2) To characterize molecularly and functionally plant innate defenses to geminivirus infection. Recently we have identified a novel layer of the plant innate defense as a virulence target of NSP from tomato- and Arabidopsis-infecting geminiviruses. We propose to identify the immediate downstream components of the transmembrane receptor NIK through protein-protein interaction approaches and reverse genetics in the model system Arabidopsis. Constitutive activation of the pathway will be achieved by mutagenesis of the receptor NIK to obtain a constitutively active kinase. Under these conditions we can analyze global changes on gene expression associated with activation of the signaling pathway as the ultimate antiviral response. We also propose to characterize AV2 from a new species of geminivirus recently found in Brazil and to examine whether this protein would suppress gene silencing or RNA interference mechanisms.

3) To examine geminivirus biodiversity in the Brazilian territory. Recent results from our group have demonstrated the extent of geminivirus diversity in Brazil. From the sequencing data of several new species of tomato-infecting geminivirus we detected recombination events among DNA-A components. Mass sequencing of tomato infecting geminiviruses, characterization of new species, and the study of recombination-adaptation-evolution aspects are the major scope of this specific goal. Because some of the recombinant progenies exhibit different pathological properties as compared to their predecessors, we are in unique position to examine the relevance of their recombinogenic properties in geminivirus evolution and emergence as agriculturally relevant pathogens.

4) To obtain tomato plants resistant to geminiviruses and to develop geminivirus-based silencing vectors. We have recently shown that dsRNA-mediated viral gene silencing was effective to confer BGMV resistance in common bean transgenic lines. We propose to exploit the same RNA interference approach to engineer broad resistance in tomato transgenic lines. We will also determine whether manipulation of the NIK-mediated antiviral signaling can increase tolerance to geminivirus and whether this pathway prevents other viruses from infecting tomato plants. Moreover, we will carry out functional genomics and proteomics study of tomato lines carrying different genes that confer resistance to geminiviruses.

5) To identify rust secreted proteins that are triggers of plant resistance and their host targets. To overcome host innate immunity, rust pathogens secrete numerous proteins (effectors) to establish disease on their hosts. In order to survive, plants have developed an advanced and highly specific surveillance system to detect these effectors and trigger disease resistance. A catalogue of putative secreted protein from P.psidii, H. vastatrix and P. pachyrhizi is being developed by The Minas Gerais Genomic Network. We will employ a range of delivery systems and assays to reveal the roles of these secreted proteins in triggering or suppressing host defense. Some of these newly found effectors will be use to determine the recognition specificity of resistance genes. Yeast two-hybrid screens will be employed to identify host targets of Avr proteins. The roles of targets in plant defense will be assessed using gene silencing techniques.

6) To assess the balance between the two alternative pathways derived from the lypoxygenase pathway and their role in mediating insect-plant interactions aiming to establish management tactics against pest insects. Soybean is the main focus of attention, and the velvetbean caterpillar (Anticarsia gemmatalis) is the main insect species under investigation. However, the effort will also be expanded to coffee plants, legume and cereal seeds, and eucalyptus using suitable pest species. The insect response to these alternative plant defense pathways will also be assessed, as well as the risk assessment of genetic suppression of the lipoxygenase pathway in soybean seeds. The purification of gut proteases from the velvetbean caterpillar will be carried out for subsequent sequencing using MALDI-TOF. The active center of the enzymes will be mapped using synthetic peptides. Synthetic protease inhibitors will be modeled to target protease inhibition on the key pest species minimizing its potential non-target impacts, which will also be assessed.

This work is directly relevant to long-range improvement and sustainability of Brazilian agriculture. Particularly in Brazil, tomato-infecting geminiviruses have emerged as economically relevant pathogens and nowadays represent permanent constraints to crop productivity. Likewise, rust fungi and phytophagous insects cause major losses in soybean. Our results will provide the identification and cloning of economically relevant insect attack-responsive genes, and fungal defense-response genes from soybean. Furthermore, key interactions between geminiviruses and their hosts will be identified to derive molecular strategies for engineering geminivirus resistance. From a biotechnological standpoint, determining and isolating genes which dictate biotic stress responses constitutes a prerequisite for understanding and modifying adaptive processes.

From a scientific point of view, the results of our program will greatly advance our knowledge about conserved as well as unique defense signaling pathways found in higher plants. Furthermore, because geminiviruses rely on both viral proteins and host factors for intra- and inter-cellular transport of viral DNA, our approach holds up the potential for the establishment of a framework for unraveling the plant nucleocytoplasmic transport machinery as well as plasmodesmata regulation for cell-to-cell transport of macromolecules.